Genes constitute only a small proportion of the total mammalian genome, and the precise control of their expression in the presence of an overwhelming background of noncoding desoxyribonucleic acid (DNA) presents a substantial problem for their regulation. Noncoding DNA, containing introns, repetitive elements, and potentially active transposable elements require effective mechanisms for its long term silencing. Mammals appear to have taken advantage of the possibilities afforded by cytosine methylation to provide a heritable mechanism for altering DNA-protein interactions to assist in such silencing. DNA methylation is essential for the development of mammals; and plays a potential role during aging and cancer. The involvement of methylation in the regulation of gene expression and as an epigenetic modification marking imprinted genes is well established. In mammals, methylation occurs only at cytosine residues and more specifically only on cytosine residues adjacent to a guanosine residue, i.e. at the sequence CG. The detection and mapping of DNA methylation sites are essential steps towards understanding the molecular signals which indicate whether a given sequence is methylated.
This is currently accomplished by the so-called bisulfite method described by Frommer, M., et al., Proc Natl Acad Sci USA 89 (1992) 1827-31 for the detection of 5-methyl-cytosines. The bisulfite method of mapping 5-methylcytosine uses the effect that sodium hydrogen sulfite reacts with cytosine but not or only poorly with 5-methyl-cytosine. Cytosine reacts with bisulfite to form a sulfonated cytosine reaction intermediate being prone to deamination resulting in a sulfonated uracil which can be desulfonated to uracil under alkaline conditions. It is common knowledge that uracil has the base pairing behavior of thymine different to the educt cytosine whereas 5-methylcytosine has the base pairing behavior of cytosine. This makes the discrimination of methylated or non-methylated cytosines possible by e.g. bisulfite genomic sequencing (Grigg, G., and Clark, S., Bioessays 16 (1994) 431-6; Grigg, G. W., DNA Seq 6 (1996) 189-98) or methylation specific PCR (MSP) disclosed in U.S. Pat. No. 5,786,146.
There are various documents addressing specific aspects of the bisulfite reaction (Benyajati, C., et al., Nucleic Acids Res 8 (1980) 5649-67) make general investigations to the bisulfite modification of 5-methyl-deoxycytosine and deoxycytosine (Olek, A., et al., Nucleic Acids Res 24 (1996) 5064-6) disclose a method for bisulfite base sequencing whereby bisulfite treatment and subsequent PCR steps are performed on material embedded in agarose beads. In the bisulfite method as disclosed by Clark, S. J., et al., Nucleic Acids Res 22 (1994) 2990-7, the sample is desalted after deamination.
Raizis, A. M., et al., Anal Biochem 226 (1995) 161-6 disclose a bisulfite method of 5-methylcytosine mapping that minimizes template degradation. They investigate the influence of pH, temperature and time of reaction. Similar investigations have been made by Grunau, C., et al., Nucleic Acids Res 29 (2001) 13e65:1-7 or Warnecke, P. M., et al., Methods 27 (2002) 101-7. Different additional components in the bisulfite mixture are disclosed by WO 01/98528 or by Paulin, R., et al., Nucleic Acids Res 26 (1998) 5009-10. An additional bisulfite step after bisulfite treatment and PCR is disclosed in WO 02/31186. Komiyama, M., and Oshima, S., Tetrahedron Letters 35 (1994) 8185-8188) investigate the catalysis of bisulfite-induced deamination of cytosine in oligodeoxyribonucleotides.
Kits for performing bisulfite treatments are commercially available from Intergen, distributed by Serologicals Corporation, Norcross, Ga., USA, e.g. CpGenome™ DNA modification kit or CpGenome™ Fast DNA Modification Kit. Another commercial kit is the EZ DNA-Methylation-Gold Kit available from Zymo Research Inc., Orange, Calif., USA. These kits provide sequential procedures comprising denaturation of purified DNA or crude lysate, deamination of DNA, desalting (=washing with a buffer that ensures the DNA to be bound to the solid phase) of deaminated DNA after binding of DNA to a solid phase, desulfonation of bound desaminated DNA by adding of alkaline, washing the DNA to remove alkaline and elution of DNA with a neutral buffer. The eluate or part of it is then usually used for PCR.
Another variation of the bisulfite genomic sequencing method is disclosed by Feil, R., et al., Nucleic Acids Res 22 (1994) 695-6, whereby the genomic DNA is bound to glass beads after deamination and washed. After elution the nucleic acid is desulfonated. It is known that nucleic acids can be isolated by the use of their binding behavior to glass surfaces, e.g. adsorption to silica gel or diatomic earths, adsorption to magnetic glass particles (MGPs) or organo silane particles under chaotropic conditions. Extraction using solid phases usually contains the steps of adding the solution with the nucleic acids to the solid phase under conditions allowing binding of the substance of interest to the solid phase, removal of the remainder of the solution from the solid phase bound nucleic acids and subsequent release of the nucleic acids from the solid phase into a liquid eluate (sometimes called elution). The result of such process is usually a solution containing the substance of interest in dissolved state.
Still another variation of the bilsulfite method is disclosed by US 2004/0241704 or EP 1 394 173. Therein a bisulfite treatment is disclosed wherein the nucleic acid is bound to a solid phase during all or only some of the steps of the bisulfite reaction.